PROJECT SUMMARY/ABSTRACT: A fundamental gap exists in our knowledge of the immunologic tumoral microenvironment during the development of acute myeloid leukemia (AML) and other deadly childhood cancers. Bridging of this gap could lead to novel immunotherapies. Our long-term goals are to develop novel immunotherapies for leukemia and pediatric solid tumors. Our objective in this application is to investigate the role of Foxp3+ regulatory T cells (Tregs) expressing Stimulation-2 (ST2), the IL-33 receptor in the microenvironment as well as ST2 on tumoral cells. Our central hypothesis is that we can target the tumoral microenvironment and tumoral cells by blocking the IL-33/ST2 pathway with neutralizing and bispecific antibodies that we will develop. This hypothesis was formed based on our unpublished preliminary data showing that: First, nonmalignant mice deficient in T-bet have elevated numbers of BM-infiltrating ST2+ Tregs compared to wild-type (WT) mice (45% vs 25%, respectively), suggesting reciprocal roles of ST2/IL33 and T- bet/interferon-? (IFN-?) in Tregs from the BM niche. Second, we observed 10-fold more Tregs expressing significantly more activation markers in the BM niche of AML-bearing mice than in the nonmalignant niche. Third, MLL-AF9 AML cell proliferation was significantly lower in mice receiving donor syngeneic ST2 knock-out (ST2-/-) T cells than in mice receiving donor syngeneic WT T cells (2% vs 17%, respectively). ST2 blockade also decreased Treg activation (i.e., KLRG1) and increased type 1 signaling in CD8+ T cells as well as decreased their exhaustion, suggesting restoration of an antitumoral response in ST2-/- mice. The rationale for this study is that once we are able to understand the biology of the immunological microenvironment in the malignant niche, we can propose personalized treatment plans to block the tolerogenic pathways. Similarly, understanding the biology responsible for the overexpression of ST2/IL-33 on the tumoral cell, will help target this pathway. This hypothesis will be tested with three specific aims: 1) Explore ST2 and IL-33 expression on tumoral cells and in the tumoral microenvironment from liquid and solid childhood cancers in human and mice; 2) Examine whether ST2/IL-33 blockade can impact tumor immunity in the malignant BM niche and solid tumor microenvironment as a proof-of-principle of a novel antitumoral immunotherapy; and 3) Optimizing anti-ST2 neutralizing antibodies against murine and human targets for translational purpose. This approach is innovative because, to our knowledge, the function of ST2+ Tregs in the malignant microenvironment, remains virtually unexplored. It will also provide biological insights into the nature of ST2+ Tregs and ST2+ tumoral cells in the microenvironment and determine whether their blockade can restore antitumoral activity and decrease tumoral proliferation via a dual mechanism. The proposed research is significant because novel antitumoral immunotherapies that are more targeted and less toxic than classical chemotherapy/irradiation regimens are needed.